The centralized hospital laboratory and the large reference laboratory remain the sites at which most clinical diagnostic testing, especially immunoassay testing, is done. There has been a convergence by the major manufacturers of immunoassay tests toward certain immunoassay formats. Among these, the sandwich immunoassay, employing both a trapping antibody and a detection antibody is the format of choice for analytes and biomarkers containing two separate epitopes to which antibodies can bind. There has been a further convergence by many leading immunoassay manufacturers on the use of antibody coupled magnetic beads as a separation means to enable the removal of the trapping antibody:antigen:detection antibody sandwich from unbound detection antibody. Magnetic beads have gained increasing use as a convenient separation technique for many forms of cell, nucleic acid and protein isolations and analyses. In particular, the manufacturers of clinical immunoassays utilize magnetic beads both as a solid support for antibodies targeted to analytes of clinical importance, and as the separation means to isolate and detect those bound analytes. Sandwich immunoassays, the most common immunoassay format, utilize two different antibodies—a trapping antibody (Ab) and a detection Ab—to form sandwich complexes in the presence of antigens. Magnetic microbeads provide an effective way to immobilize sandwich complexes while washing away unbound detection Ab. But use of magnetic beads imposes a paradox. The magnetic beads must have sufficient size—normally on the order of several microns in diameter—in order to be separated in an easily achievable magnetic field. But magnetic beads of this size diffuse only very slowly, and present very limited surface area for antibody binding compared to their volume. Thus the size of the current magnetic beads limits the speed and sensitivity that can be achieved in clinical immunoassays.
Through advances in template-directed polymer synthesis and nanotechnology, a new class of “smart” magnetic nanoparticles can be made. These magnetic nanoparticles can change from a monodispersed small diameter particle of roughly 20 nanometers diameter to a macro-aggregate of microns diameter in response to an environmental stimulus like a temperature or pH change. By using these advanced nanomaterials, assays can be developed in which the very high surface to volume ratio and the small size/high diffusion of the smart magnetic nanoparticles provides for higher sensitivity (greater antigen binding) and faster binding reactions compared to current magnetic bead reagents. A discrete pH or temperature stimulus can cause these smart nanoparticle reagents to co-aggregate with other entities containing stimuli-responsive polymers into a macro-aggregate of micron dimensions, which can be separated by a magnetic field as easily and quickly as currently used magnetic beads.
These new to the world materials present the promise of faster, more sensitive clinical immunoassays for important biomarkers of cardiac disease, cancer, endocrine and infectious diseases. These smart magnetic nanoparticles can show the same advantages in life science research applications currently using magnetic beads for the separation or analysis of cells, proteins, or nucleic acid sequences. The present invention seeks to fulfill this need and provides further related advantages.